The increasing application of recombinant DNA technology to engineer novel microorganisms which are industrially useful have caused concerns in both the scientific community and the general public over potential risks. These concerns are primarily related to the potential harm to humans and to undesirable and/or uncontrollable ecological consequences upon deliberate or unintentional release of such genetically engineered microorganisms (GEMs) into the environment. These concerns have led to the establishment of official guidelines for the safe handling of GEMs in laboratories and production facilities where such organisms are applied. Up till now, such guidelines have primarily been directed to measures of physically containing GEMs in laboratories and production facilities with the aim of reducing the likelihood that workers in such facilities were contaminated, or that the GEMs were to escape from their primary physical environment, such as a fermentation vessel.
It is presently being recognized that the level of safety in the handling of GEMs can be increased by combining physical containment measures with biological containment measures to reduce the possibility of the survival of the genetically engineered organisms if they were to escape from their primary environment.
Lately, however, concerns have become increasingly focused on potential risks related to deliberate release of GEMs to the outer environment and to the use of GEMs as live vaccines. In this connection there is a strongly felt need to have biological containment systems which subsequent to the environmental mental release of the GEMs or their administration as vaccines to a human or an animal body, effectively kill the released organisms in a controlled way or which limit the function of the released GEMs to an extent where such GEMs are placed at a significant competitive disadvantage whereby they will eventually be ousted by the natural microflora of the environment to which they are released.
The first systems of biological containment were based on the use of "safe" cloning vectors and debilitated host bacteria. As examples, it has been suggested to select vectors which lack transfer functions or which naturally have a very narrow host range. Examples of debilitated host bacteria are E. coli mutants having an obligate requirement for exogenous nutrients not present or present in low concentrations outside the primary environment of the GEMs.
Other suggested biological containment systems have been based on mechanisms whereby the vector is restricted to the GEMs e.g. by using a plasmid vector with a non-sense mutation in a gene, the expression of which is indispensable for plasmid replication, or a suppressor mutation in the chromosome, said mutation blocking translational read-through of the message of the gene. A further approach is to maintain the rDNA stably in the host by integrating it into the chromosomes of the GEMs.
Recently, an alternative biological containment strategy has been developed in which the recombinant vector is endowed with a gene encoding a cell killing function which gene is under the control of a promoter only being expressed under certain environmental conditions, such as conditions prevailing in an environment outside the primary environment of the GEMs, or when the vector is unintentionally transferred to a secondary host, or the expression of which is stochastically induced. By using incorporation in a GEM of such a cell killing function and selecting appropriate regulatory sequences, vectors can be constructed which are contained in the primary host cell and/or in a primary physical environment. A cell killing function as hereindefined may also be referred to as an active biological containment factor.
If a stochastically induced mechanism of expression regulation is selected for such a biological containment system, a population of GEMs containing the system will, upon release to the outer environment, or if used as a live vaccine, be subjected to a random cell killing which will lead to an increase of the doubling time of the host cell population or eventually to the disappearance of the organisms.
The above-mentioned genes encoding cell killing functions are also frequently referred to as "suicide" genes, and biological containment systems based upon the use of such genes, the expression of which are regulated as defined above, are commonly described as conditional lethal systems or "suicide" systems. Up till now, several cell killing functions have been found in bacterial chromosomes and in prokaryotic plasmids. Examples of chromosomal genes having cell killing functions are the gef (Poulsen et al., 1991) and relF (Bech et al., 1985) genes from E. coli K-12. Examples of plasmid encoded suicide genes are hok and flmA (Gerdes et al., 1986) genes isolated from plasmids R1 and F, respectively, the snrB gene also isolated from plasmid F (Akimoto et al., 1986) and the pnd gene isolated from plasmids R16 and R483 (Sakikawa et al., 1989 and Ono et al., 1987). Common features of these genes are that they are transcribed constitutively, regulated at a post-transcriptional level, and that they all encode small toxic proteins of about 50 amino acids. The application of the hok gene in a biological containment system has been disclosed in WO 87/05932.
Ideally, the features of an effective biological containment system should include as a minimum requirement that the cell killing function when it is expressed, is effective, that the containment system is functional in a broad range of species of GEMs, that the risk of elimination of the cell killing function e.g. by mutations in the suicide gene or the sequences regulating the expression of the gene, is minimal and that the risk of uptake by other organisms of rDNA released when cells are killed, is reduced.
None of the above-mentioned known containment systems fulfil all of these ideal requirements. However, the present invention provides a novel active biological containment system which is not based on a primary cell killing function but which makes use of genes, the expression of which in a cell where the gene is inserted, results in the formation of mature forms of exoenzymes which are hydrolytically active in the cytoplasm of the cell and which can not be transported over the cell membrane. When such enzymes are expressed, the normal function of the cell becomes limited to an extent whereby the competitiveness, and hence the survival, of a population of such cells is reduced significantly.
In this connection, it has been observed that although an exoenzyme such as a bacterial nuclease encoded by a gene from which the sequence coding for the signal peptide has been totally removed may be entirely suitable in a method of limiting the survival of GEMs in accordance with the above, a certain transport of the nuclease out of the cell comprising such a gene without the signal sequence-coding sequence may cause "leakage" of active enzyme out of the cell, thereby reducing the efficiency of this containment system. However, it has surprisingly been found that this problem can be overcome if a truncated and/or mutated form of a nuclease is used. Additionally, a significant increase in the cytoplasmic enzymatic activity of such a modified enzyme relative to a parent nuclease has been provided thus resulting in further improved methods of limiting the survival of GEMs. Accordingly, the hydrolytically active enzyme of the invention includes such a truncated and/or mutated form of a nuclease.
Provided the hydrolytically active enzyme is an RNA-degrading and/or DNA-degrading enzyme, a biological containment system based upon such an enzyme may have a further advantage over the known biological containment systems in that the rDNA molecules in a genetically engineered host cell is destroyed simultaneously with the genetically altered host microorganisms.
As an example, a recombinant staphylococcal nuclease according the invention having endonucleolytic activity will when used in accordance with the invention cause hydrolysis of the DNA and the RNA to 3'-phosphomononucleotides. However, it may from a GEM risk point of view be advantageous to provide the GEM containment system in a form where further intracellular degradation of the damaged nucleic acids is provided. This may e.g. be provided by inserting into the GEMs a further gene coding for a 3'-5' exonucleolytically active exonuclease. The simultaneous expression and the successive degradative activities of a endonuclease such as the above modified staphylococcal nuclease and an exonuclease will cause complete destruction of the host nucleic acids, thus killing the host strain without having its nucleic acids remaining stable in the environment for an unlimited period of time.